Technical Field
The present invention relates to a surface-plasmon enhanced fluorescence measurement method and a surface-plasmon enhanced fluorescence measurement device for detecting a detection-target substance contained in a sample solution, using surface plasmon resonance (SPR), and also relates to an analysis chip used in detection of the detection-target substance contained in the sample solution.
Background Art
Highly-sensitive and quantitative detection of a slight amount of a detection-target substance in measurement for detecting biological substances such as protein and DNA makes it possible to immediately figure out the condition of a patient and treat the patient. For this reason, there has been a demand for an analysis method and an analysis device for highly-sensitive and quantitative detection of weak light caused by a slight amount of the detection-target substance. As an exemplary method of detecting a detection-target substance with high sensitivity, a surface plasmon resonance fluorescence analysis (Surface Plasmon-field enhanced Fluorescence Spectroscopy (SPFS)) is known.
SPFS uses a prism provided with a metal film disposed on a predetermined surface of the prism. Irradiation of the metal film with excitation light through the prism at an angle at which surface plasmon resonance occurs can generate localized light (enhanced electric field) on the surface of the metal film. This localized light excites a fluorescent substance used for labeling the detection-target substance captured on the metal film, therefore making it possible to detect the presence or amount of the detection-target substance through detection of the fluorescence emitted from the fluorescent substance.
In SPFS, highly-sensitive and accurate detection requires accurate positioning of the analysis chip. Accurate detection of the amount (density) of the detection-target substance requires accurate adjustment of the incident angle of excitation light. However, when the analysis chip is shifted in position, accurate adjustment of the incident angle of the excitation light is impossible. In addition, the irradiation spot of the excitation light and the reaction site on the metal film preferably coincide with each other in shape and position for highly-sensitive detection of the detection-target substance. However, when the analysis chip is shifted in position, the irradiation spot of excitation light cannot be adjusted accurately in shape and position. Meanwhile, requiring users to accurately position the analysis chip is unfavorable in terms of usability.
There have been proposed methods for positioning an analysis chip in methods for detecting a detection-target substance by irradiating the analysis chip with light although they are not SPFS. For example, Patent Literature (hereinafter, referred to as “PTL”) 1 discloses identifying the position of an analysis chip (biochip) through irradiation of the analysis chip with illumination light different from excitation light in wavelength and detection of reflection light or transmission light of the illumination light in detection using a fluorescent substance. The use of illumination light different from excitation light in wavelength makes it possible to identify the position of the analysis chip while preventing the fluorescent substance from being discolored.